NIH CRM is a Common Fund-supported initiative that was established in 2010. The efforts of Mahendra Rao, the NIH CRM Director appointed in August 2011, are split between the Center and his intramural laboratory. Since his appointment, Dr. Rao has completed recruitment of the key positions in the laboratory. This comprises two Staff Scientists, Nasir Malik and Jizhong Zou. They in turn have assisted Dr. Rao in hiring two biologists, Xiantao Wang and Yongquan Luo. The laboratory group has also included non-permanent staff in the past year, including post-doctoral and post-baccalaureate fellows. Efforts in neural development have focused on developing and optimizing protocols for the generation of differentiated cell types that can have applications in translational medicine. Several screening projects with neural cells are currently underway with collaborators at NCATS and NINDS. One of these, which was recently published (Swaroop et al., 2012), indicated that cholesterol activity could be assessed in iPSC-derived neurons and commercial fetal astrocyte. A screen in progress with NCATS is examining whether compounds that protect astrocytes from cell death can be identified. Another in-progress screen with NINDS is attempting to identify drugs that are selectively toxic to human neural stem cells (NSC). The lab also has a paper in press (Efthymiou et al., in press) demonstrating that neurons derived from NSCs are amenable to viability and high content screens in a 96-well format. The work that has been performed in this area sets the stage for screens in cells derived from patients with neurological disorders to identify new compounds that may be of therapeutic value. In addition to the screening projects the lab is also attempting to better understand the mechanisms underlying the transition from a multipotent neural stem cell stage to terminally differentiated neurons and astrocytes. Microarray analysis is being used to identify genes and signaling pathways critical in this transition and one paper has been submitted detailing this process in astrocytes (Malik et al., manuscript submitted). Ongoing work is also looking at how neural differentiation is affected in iPSC-derived neural stem cells from patients with amytrophic lateral sclerosis and Niemann-Pick type C disease (NPC). In the case of NPC, the analysis indicates premature glial differentiation and misregulation of the Wnt signaling pathway, providing a possible assay for a drug screen to identify novel compounds that could have therapeutic value. The laboratory also has an active gene engineering program focusing on human iPSCs and their derivatives. The lab has established its own open-source TALEN assembly platform. Using AAVS1 safe harbor as our first target, we compared different AAVS1-ZFN/TALEN efficiency, and by both targeted mutagenesis and homologous recombination assays, our open-source TALENs showed the highest efficiency (4-fold higher than commercial AAVS1-ZFN) in human cells. We also developed novel TALENs, targeting new safe harbor on Chromosome 13, which showed 25% gene-editing efficiency. In terms of genome engineering using human iPSCs and NSCs, we designed two sets of universal donors to accommodate various gene targeting tasks. One set is designed to target safe harbor loci such as AAVS1 on chr. 19 (AAVS1 safe harbor) or CLYBL2 on chr. 13 (C13 safe harbor). We have used these TALENs and donors to generate several reporter cell lines driven by constitutive or tissue-specific promoters. We have achieved simultaneous gene knock-in at double safe harbor alleles to engineer multiplex reporter iPSC lines. Using same genome editing TALENs and donors described above, we have achieved single-/double-safe harbor reporter knock-in in human NSCs. From this exciting work we have filed 6 EIRs on 2 pairs of safe harbor TALENs, 11 AAVS1 donors, 2 gene-specific donors, 9 safe harbor engineered human iPSC lines, and the methods and compositions of Chr. 13 safe harbor locus for targeted genome modification, which we are also filing patents on. These materials have been transferred to 32 academic labs worldwide and 10 companies through SLA, MTA or BMLA. We are also depositing three engineered human iPSC lines to Rutgers, Sigma and other cell repositories for broader distribution. Two of the companies, SystemBiosciences and Geneocopeia, have launched the commercial products based on our AAVS1 TALENs and donor vectors. We have also provided consultation and support to several dozen academic labs inside or outside NIH to learn TALEN and other genome engineering technologies through an NIH CRM-sponsored Bio-Trac training course. Collectively, these efforts in neural development and genome engineering are all part of NIH CRMs overarching goal of translating cell-based therapy to the clinic. The laboratory has maintained its focus on neural derivatives, and these initial reports showcase an efficient process for manufacture of iPSC and engineering of pluripotent cells, which brings us one step closer to clinical translation. Publications Submitted/Accepted during 2013 Reporting Period: 1. Cerbini, T., R. Funahashi, et al. (Submitted). Multiplexed gene-targeting at safe-harbors in human iPSCs and neural stem cells. Nature Methods. 2. Efthymiou, A., A. Shaltouki, et al. (Accepted). Functional Screening Assays with Neurons Generated from Pluripotent Stem Cell-Derived Neural Stem Cells. J Biomol Screen. 3. Luo, Y., C. F. Liu, et al. (Submitted). Stable EGFP expressing after differentiation and transpantation of reporter human iPSCs generated by AAVS1-TALENs. Stem Cells. 4. Malik, N., Shah, S., Efthymiou, A., Yan, B., Zhan, M., and Rao, M. (Submitted). Comparison of the gene expression profiles of human fetal cortical astrocytes with pluripotent stem cell derived neural stem cells identifies human astrocyte markers and signaling pathways and transcription factors active in human astrocytes. PLoS One. 5. Malik, N., S. Shin, et al. (Submitted). Genomic Analyses of Neural Stem Cells. Principles of Developmental Genetics, 2nd Edition. S. Moody. Elsevier. 6. Rao, M. and N. Malik (Submitted). Neural Differentiation of Pluripotent Stem Cells. Stem Cells Handbook. S. Sell, Springer. 7. Yuan, X., E. M. Braunstein, et al. (Accepted). Generation of GPI Anchor Protein Deficient Blood Cells From Human Induced Pluripotent Stem Cells. Stem Cell Translational Medicine. 8. Zou, J. and L. Cheng (2013). Advanced Textbook on Gene Transfer, Gene Therapy and Genetic Pharmacology. Chapter 25., Imperial College Press.